Novel substrates for use in measuring the concentration of kallikrein in urine

ABSTRACT

A novel chromogenic and fluorescent substrate for measuring the concentration of kallikrein in urine. The novel substrate according to the present invention has very excellent substrate specificity to kallikreins and good solubility in water or biological test solutions. The present substrate is therefore useful for measuring the concentration of kallikrein in urine for the diagnosis of hypertension and other diseases caused by the lowering of the concentration of kallikrein.

FIELD OF THE INVENTION

The present invention relates to novel chromogenic and fluorescentsubstrates for use in measuring the concentration of kallikrein inurine.

Novel substrates for use in measuring the concentration of kallikrein inurine according to the present invention are represented by the generalformula (I), ##STR1## [wherein A₁ is Pro (prolyl group), PGlu(pyroglutamyl group), Val (valyl group), Ala (alanyl group), Leu (leucylgroup), Phe (phenylalanyl group) or Lys (lysyl group)].

The novel substrates according to the present invention haveconsiderably higher selectivity to kallikrein as compared with anysubstrate being used in measuring the concentration thereof known inprior art literatures, and thus the substrates according to the presentinvention can be able to measure the concentration of kallikrein inurine with higher sensitivity.

Kallikrein is enzyme which converts kininogen in plasma into kinin whichis pharmacologically active peptide. Biological activities of kallikreinwhich are performed through the kinin being converted therefrom areactivities in increasing for example vasodilation and hypotension or thelike.

At the beginning of studies, kallikrein in human urine have been thoughtas originated in the pancreas. While, recently, they have been thoughtas originated in the kidney, and thus much attention have been paid totheir roles in the renal functions, particularly, it is believed thatthey are related to control the renal blood flow rate and to adjust theblood pressure. Therefore, decreasing of the biosynthesis and of thesecretion of kallikrein will cause serious diseases. For example, theconcentration of kallikrein in urine of the patients of essentialhypertension and of renal hypertension is considerably lowered ascompared with that of a normal healthy person. Additionally, it is knownthat the concentration of kallikrein in urine of the patients of primaryaldosteronism and of Bartter syndrome is higher, while the concentrationof kallikrein in urine of the patients of renal failure is lowered.

Therefore, measurements of kallikrein in urines of the patients ofhypertension and of other diseases are quite important and useful fordiagnosis of such disease.

DESCRIPTION OF THE PRIOR ART AND THE PROBLEMS INVOLVED

Methods for determining the activities of kallikrein in human urine havebeen reported in a number of literatures. However, there have beenraised a number of problems in such methods due to their complexity inoperations, the influences of inhibitory substances exist in urine andother factors involved therein.

According to the developments in chemistry of synthetic substrates madein recent years, some of methods for determining the activities ofkallikrein in human urine have been improved in certain extents. Forexample, a method for determining the hydrolytic activity of kallikreinagainst synthetic substrates such as TAME (Tos-Arg-Ome), BAFE(Bz-Arg-OEt), TLME (Tos-Lys-OMe) or the like is, one hand simple in theoperation as well as higher repeatability, but on the other hand low inthe specificity of the substrates and the sensitivity.

Furthermore, as to the substrate for determining the activities inkallikrein in human urine, there have been developed H-D-Val-Leu-Arg-PNA(S-2266: Kabi), by G. Claeson et al., "Haemostasis, Vol. 7, page 62(1978)". Similarly, there have also been developed fluorescent peptideof H-Pro-Phe-Arg-MCA (methyl cumarinamide) by coupling with AMC(amino-methylcumarin) which will emit the fluorescence when it isliberated therefrom, by T. Morita et al., "J. Biochem. (Tokyo), Vol. 82,page 1495 (1977)".

On the other hand, as to the synthetic substrate for use indetermination of the activity of the enzyme, it is important to satisfythe following four points: (1) high sensitivity to the enzyme to bedetermined, (2) high specificity to the enzyme to be determined, (3)good solubility in water or biological test solutions, and (4) highdetectability for the decomposed substrates. Among these four points,high specificity to the enzyme to be determined is the most important.

In the case of determination of the activity of kallikrein in urine byusing chromogenic substrate, the result of determination with highaccuracy cannot be expected when interactions were occurred between thesubstrate and proteolytic enzymes such as plasmin, thrombin, urokinaseor the like, other than the kallikrein, which may be existed in theurine. In this connection, it should be noted the fact that, theabove-mentioned S-2266 which was developed as the best substrate for usein the determination of kallikrein is not considered as a substratesatisfying the specificity as well as the reactivity. Thus, theabove-mentioned substrate of S-2266 may have some dangers to give excessdetermination results caused by α-thrombin and plasmin which areappearing sometimes in abnormal urine to be examined. Additionally,influences of hydrolysis being caused by urokinase secreted in urineshould also be considered when the above-mentioned substrate S-2266 isused.

In addition to the above, in conducting a colorimetry by using asubstrate which may be produce yellow color of p-nitroaniline separatedfrom the substrate similar to the above-mentioned substrate of S-2266,the test results obtained therefrom cannot be escaped from theinfluences of the color of urine sample to be examined.

Furthermore, in conducting a fluorescence method, the test result shouldbe corrected by conducting a blank test for substracting the influencescaused by fluorescent substances which may be existed in the urinesample to be examine.

In order to improve such drawbacks shown in conventional substrates, thepresent inventors have made extensive research and development works forfinding novel substrates for use in determination of kallikrein inurine, and as the results, the present inventors have find novelsubstrates which were improved from the above-mentioned drawbacks aswell as possess excellent properties satisfying the above-mentionedessential four conditions.

Thus, the present inventors have succeeded in developing novelsubstrates for use in determination of kallikdrein in urine of whichselectivity was considerably improved as compared with that ofconventional substrates of which reactivities against enzymes other thanthe objective kallikrein in urine, such as plasmin, thrombin, urokinaseor the like being inhibited by using 3-carboxy-4-hydroxyanilide(hereinafter referred to as "CHA") in place of p-nitroanilide(hereinafter referred to as "PNA") which was used as the chromogenicgroup in the substrates being developed before.

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel chromogenic andfluorescent substrates for use in measuring the concentration ofkallikrein in urine with higher sensitivity.

Another object of the present invention will be apparent from thedescriptions below in this specification.

DETAILED EXPLANATION OF THE INVENTION

Novel chromogenic and fluorescent substrates for use in measuring theconcentration of kallikrein in urine according to the present inventionare represented by the general formula (I) as follows: ##STR2## [whereinA₁ is Pro (prolyl group), PGlu (pyroglutamyl group), Val (valyl group),Ala (alanyl group), Leu (leucyl group), Phe (phenylalanyl group) or Lys(lysyl group)], specifically the substrate is characterized by the useof 3-carboxy-4-hydroxyaniline (CHA) as the chromogenic group. Thepresent substrate has excellent solubility in water because of thepossession of extremely hydrophylic groups, i.e., hydroxyl and carboxylgroups as the chromogenic groups. In a typical examples of practical useas a substrate for the determination of kallikrein in urine, the formed3-carboxy-4-hydroxyaniline is converted into a colored substance byusing a pentacyanoamineferroate method or by an oxydative condensationwith a suitable coupler, said colored substance being then subjected toa colorimetric determination. It is also possible to determine theactivity of kallikrein in urine specifically by making fluorometricdeterminations at excitation wavelength of 328 nm and fluorescentwavelength of 540 nm.

The salient features of the present substrate, as stated above, residein its excellent substrate specificity to kallikrein in urine. Asregards to the substrate specificity, the relative reactivity of thepresent novel substrate PS-2103 (H-D-Ala-Phe-Arg-CHA) in variousproteases in urine, that is, kallikrein (HUK), thrombin (TH), plasmin(PL), urokinase (UK) and others is shown in the following Table 1 alongwith those of PS-2103N (H-D-Ala-Phe-Arg-PNA) and S-2266 which weresynthesized as to reference compound by way of using PNA as thechromogenic group with the same amino acid configurations as said novelsubstrate. In Table 1, the relative reactivity of PS-2103N(H-D-Ala-Phe-Arg-PNA) in each of said enzymes is given as 100 forcriterion. As can be seen from Table 1, the relative reactivity toplasmin, thrombin and urokinase is extremely low in the novel substrateusing CHA as the chromogenic group. It shows only 4% reactivity toplasmin, 7% reactivity to thrombin and 0% reactivity to urokinase. It isalso noted that the novel substrate is markedly improved in itsselectivity in comparison with S-2266 which shows 54% reactivity toplasmin, 138% reactivity to thrombin and 217% reactivity to urokinase,specifically, the present novel substrate does not react with urokinase.

The above-mentioned facts indicate that the novel substrate according tothe present invention is excellent in determination of kallikrein inurine.

                  TABLE 1                                                         ______________________________________                                        Relative reactivity to enzymes                                                Substrate tested HUK     TH      PL    UK                                     ______________________________________                                        S-2266           225     138     54    217                                    H--D-Val--Leu--Arg--PNA                                                                        (0.245) (0.065) (0.067)                                                                             (0.013)                                PS-2103N         100     100     100   100                                    H--D-Ala--Phe--Arg--PNA                                                                        (0.109) (0.047) (0.125)                                                                             (0.006)                                PS-2103 (Substrate of                                                                          242     7       4     0                                      the present invention)                                                                         (0.599) (0.007) (0.011)                                                                             (0.0)                                  H--D-Ala--Phe--Arg--CHA                                                       ______________________________________                                         Initial substrate concentration S.sub.0 = 0.4 m mol. Figures in the           parentheses are determined OD (optical density) values.                       Determinations were made at wavelength of 405 nm in S2266 and PS2103N, an     700 nm in PS2103.                                                        

The primary use of the compound of the present invention is a substratefor determining the activity of kallikrein in urine as already stated.In this case, the substrate is acted to kallikrein in urine in a buffersolution having a pH of 8.5-9.0 and the formed 3-carboxy-hydroxyanilineis led into a proper colored substance, and this colored substance issubjected to colorimetric determinations so as to determine the activityof kallikrein in urine. Furthermore, fluorometric determination at anexcitation wavelength of 328 nm and a fluorescent wavelength of 540 nmis also possible.

As to the method for leading said reaction product(3-carboxy-4-hydroxyaniline) into a colored substance, apentacyanoamineferroate method or a method in which said reactionproduct is subjected to oxidative condensation with a coupler. As to thecoupler, an aniline compound, for example, N,N-diethylaniline can beused in case of color development in an acidic condition, and phenol,naphthol, thymol, o-cresol, o-ethylphenol and the like can be used forthe color development in an alkaline condition. A variety of oxidizingagents such as hydrogen peroxide, persulfate and the like can be used asthe oxidizing agents for oxidative condensation, and particularlymetaperiodic acid is perferred.

By the virtue of transformation of 3-carboxy-4-hydroxyaniline into aproper colored substance, the maximum wavelength distribution isconfined in the range of 560-770 nm and the variation of coloration dueto temperature is minimized and stabilized to provide a situation suitedfor the determination of the activity of kallikrein in urine. Asignificant difference in chromogenic sensitivity is also noted. In thecase of p-nitroaniline, ε=10,600 at ordinary measuring wavelength of 405nm, whereas in the pentacyanoamineferroate method, ε=21,500 at λ=700 nm,and in color development by oxidative condensation, in case ofo-ethylphenol, ε=29,000 at λ=645 nm, and in case of 2,6-xylenol,ε=21,600 at λ=615 nm. Such high absorbance proves to be greatly helpfulfor making determinations.

A prominent advantage of the present invention is that thedeterminations are scarcely affected by the impurities in the urinesample. In the case of p-nitroaniline compounds, determinations are madeat a wavelength below 560 nm while according to the present invention,determinations are conducted at a wavelength above 560 nm, so thatdeterminations are kept free of influence by the impurities in thesample, and this, coupled with the intrinsically high specificity of thesubstrate, makes it possible to obtain a very accurate result ofdetermination.

From the foredoing description, it will be apparent that the compound ofthe present invention can provide a very excellent substrate to be usedfor determining the activity of kallikrein as compared with theconventional ones.

The compound of the present invention represented by the general formula(I) can be synthesized by a method well known in peptide chemistry.

As to the α-amino protecting group, it is advantageous to usecarbobenzoxy group or t-butyloxycarbonyl group, or the groups relatedthereto such as p-methoxycarbobenzoxy group, p-nitrocarbobenzoxy groupor p-methoxyphenylazolecarbobenzoxy group or the like.

For the protection of γ-guanidyl group of arginine, it is advantageousto use nitro group or protonization means. For the coupling of two aminoacids or coupling of α-carboxyl group. For such coupling,N-hydroxysuccinimide, p-nitrophenol, trichlorophenol,4,6-dimethylpyrimidyl-2-thiol and the like can be used. Above-mentionedactivation into an ester derivative is advantageously conducted in thepresence of a carbodiimide such as N,N-dicyclohexylcarbodiimide (DCC).

For the synthesis of the substrate, a method is used in whichchromogenic group is first bonded to arginyl group, followed bysuccessive coupling. Alternatively, N-terminated dipeptide fragmentitself may be first synthesized and it is then bonded to arginyl grouphaving chromogenic group.

The present invention will be described in further detail below by wayof the embodiments thereof, however it is to be understood that thepresent invention is not limited only to the scope of these embodiments.

Notice is to be taken of the following matters in reading through theExamples given below.

(1) Abbreviations:

Arg=arginyl

Phe=phenylalanyl

Pro=prolyl

PGlu=pyroglutamyl acid

Val=valyl

Ala=alanyl

Leu=Leucyl

Lys=lysyl

Z=benzyloxycarbonyl

BOC=t-butyloxycarbonyl

DMF=dimethylformamide

MeOH=methanol

NEM=N-ethylmorpholine

--PNA=p-nitroanilide

--CHA=3-carboxy-4-hydroxyanilide

TLC=thin layer chromatography

AcOH=acetic acid

BuOH=butanol

AcOEt=ethyl acetate

(Note: The amino acids are defined as all L-isomers unless otherwisenoted.)

(2) Thin layer chromatography (TLC):

Silica gel F₂₅₄ plate (manufactured by E. Merck A. G.) was used for TLCanalysis.

Solvents used in the TLC are as follows:

Rf₁ CHCl₃ :MeOH:AcOH:H₂ O=80:40:2.5:5

Rf₂ n-BuOH:AcOH:H₂ O=4:1:1

Rf₃ n-BuOH:AcOH:H₂ =4:1:5

(3) Gel filtration:

"TOYOPEARL HW 40F" a trademark for polyvinyl gel manufactured by ToyoSoda Manufacturing Co., Ltd. was used for the gel filtration.

EXAMPLE 1 Synthesis of H-D-Pro-Phe-Arg-CHA

I. BOC-Arg(NO₂)-CHA

50.3 Grams (0.158 mole) of BOC-Arg(NO₂)-OH was dissolved in 160 ml ofDMF, to this solution was added 20.5 ml (0.158 mole) of NEM, followed bydropwise addition of 21.2 ml (0.158 mole) of isobutyl chloroformate at-20° C. and reacted for 10 minutes. After the reaction, a solution of250 ml of DMF, 30.0 g (0.158 mole) of 5-amino-salicylic acidhydrochloride and 61.6 ml (0.474 mole) of NEM was added dropwise to saidreaction mixture at -15° to -10° C. After dropwise addition, thereaction was continued at the same temperature for 3 hours, thenadditionally reacted at room temperature (15° to 20° C.) for 18 hours.After the reaction, DMF was removed by evaporation under reducedpressure and the residue thus obtained was dissolved in 950 ml of AcOEt.The AcOEt solution was washed 4 times with 300 ml of cold 5%hydrochloric acid, washed twice with 300 ml of an aqueous solutionsaturated with sodium chloride, then decolored and dried with activatedcarbon and anhydrous magnesium sulfate. After drying, magnesium sulfateand activated carbon were removed by filtration, and the filtrate wascooled and allowed to stand quietly. The crystals precipitated in thefiltrate were collected by filtration and dried to obtain 46.2 g (64.3%)of BOC-Arg(NO₂)-CHA.

Rf₁ =0.11. Melting point: 208° C. (decomposed).

[α]_(D) ²⁰ -8.4 (C=1, MeOH).

    ______________________________________                                        Elementary analysis (for C.sub.18 H.sub.26 N.sub.6 O.sub.8.1/4H.sub.2 O)                 C         H      N                                                 ______________________________________                                        Calculated (%):                                                                            47.11       5.82   18.31                                         Found (%):   47.16       5.75   18.30                                         ______________________________________                                    

II. BOC-Phe-Arg(NO₂)-CHA

45.4 Grams (0.1 mole) of BOC-Arg(NO₂)-CHA was dissolved in 300 ml (0.6mole) of 2N-HCl/AcOH with a small amount of MeOH, and the mixture wasreacted at room temperature for 2 hours. After the reaction wascompleted, 300 ml of dried diethyl ether was added to the reactionmixture so as to precipitate the crystals. There was obtained 34.9 g(89.4%) of H-Arg(NO₂)-CHA.HCl.

Rf₃ =0.31. Melting point: 193°-206° C.

[α]_(D) ²⁰ +37.4 (C=1, MeOH).

Next, 31.3 g (0.08 mole) of H-Arg(NO₂)-CHA.HCl was dissolved in 160 mlof 1.5N-NEM/DMF, to this solution was added 31.0 g (0.08 mole) ofBOC-Phe-SDP (dimethylmercaptopyrimidine) at 0° to 5° C. and reacted for18 hours. After the reaction was completed, the reaction mixture wasdiluted with 1600 ml of AcOEt, and washed twice with 400 ml of cold 5%hydrochloric acid, and washed twice with 400 ml of an aqueous solutionsaturated with sodium chloride, then there was precipitated crystals.The crystals thus precipitated were collected by filtration and dried toobtain crude BOC-Phe-Arg(NO₂)-CHA, then recrystallized fromAcOEt/n-hexane to obtain 35.1 g (72.9%) of BOC-Phe-Arg(NO₂)-CHA.

Rf₁ =0.91. Melting point: 212.5° C. (decomposed).

[α]_(D) ²⁰ -5.2 (C=1, DMF).

    ______________________________________                                        Elementary analysis (for C.sub.27 H.sub.35 N.sub.7 O.sub.9.H.sub.2 O)                    C         H      N                                                 ______________________________________                                        Calculated (%):                                                                            52.34       6.02   15.82                                         Found (%):   52.43       5.79   15.64                                         ______________________________________                                    

III. BOC-D-Pro-Phe-Arg(NO₂)-CHA

34.3 Grams (0.057 mole) of BOC-Phe-Arg(NO₂)-CHA was dissolved in 171 ml(0.342 Mole) of 2N HCl/AcOH with a small amount of MeOH, and reacted atroom temperature for 2 hours. After the reaction was completed, 2 litersof dried diethyl ether was added to the reaction mixture so as toprecipitate crystals. The precipitated crystals were collected byfiltration and dried to obtain 30.9 g (100%) of H-Phe-Arg(NO₂)-CHA.HCl.

Rf₂ =0.54. Melting point: 241° C. (decomposed).

[α]_(D) ²⁰ -18.0 (C=1.0, DMF).

Next, 1.34 g (2.5 m moles) of H-Phe-Arg(NO₂)-CHA was dissolved in 10 mlof 0.75N-NEM/DMF, to this solution was added 0.84 g (2.5 m moles) ofBOC-D-Pro-SDP at 0° to 5° C. and reacted at room temperature for 18hours. After the reaction was completed, the reaction mixture wasdiluted with 150 ml of AcOEt, and washed 4 times with 50 ml of cold 5%hydrochloric acid, and washed twice with 50 ml of an aqueous solutionsaturated with sodium chloride, then decolored and dried with activatedcarbon and anhydrous magnesium sulfate. After drying, magnesium sulfateand activated carbon were removed by filtration, and the solvent wasremoved from the filtrate by evaporation under reduced pressure toobtain crude product of BOC-D-Pro-Phe-Arg(NO₂)-CHA, recrystallized fromAcOEt to obtain 1.2 g (85.9%) of BOC-D-Pro-Phe-Arg(NO₂)-CHA.

Rf₁ =0.26. Melting point: 231.5° C. (decomposed).

[α]_(D) ²⁰ -2.8 (C=0.5, MeOH).

    ______________________________________                                        Elementary analysis (for C.sub.32 H.sub.42 N.sub.8 O.sub.10)                             C         H      N                                                 ______________________________________                                        Calculated (%):                                                                            55.01       6.06   16.04                                         Found (%):   54.76       6.06   15.78                                         ______________________________________                                    

IV. H-D-Pro-Phe-Arg-CHA.2HCl

0.90 Gram (1.28 m mole) of BOC-D-Pro-Phe-Arg(NO₂)-CHA was dissolved in3.8 ml of 2N-HCl/AcOH with a small amount of MeOH and reacted at roomtemperature for 2 hours. After the reaction was completed, the solventwas removed by evaporation under reduced pressure, the residue thusobtained was suspended in 113 ml of MeOH, 37 ml of H₂ O and 2.2 ml of1N-HCl. Next, 1 g of palladium black was added thereto and reduced byhydrogenation at 30° C. for 6 hours. After the hydrogenation wascompleted, the catalyst was removed by filtration and the solvent wasalso removed by evaporation under reduced pressure. The residue thusobtained was purified by using a gel filtration using TOYOPEARL HW 40Fcolumn with MeOH as the developing solvent to obtain 0.66 g (95.3%) ofH-D-Pro-Phe-Arg-CHA.2HCl.

Rf₃ =0.30. Melting point: 230° C.

[α]_(D) ²⁰ -5.0 (C=0.5, MeOH).

    ______________________________________                                        Elementary analysis (for C.sub.26 H.sub.34 N.sub.8 O.sub.5.3HCl.H.sub.2       O)                                                                                       C         H      N                                                 ______________________________________                                        Calculated (%):                                                                            46.89       5.90   16.82                                         Found (%):   47.01       5.62   16.88                                         ______________________________________                                    

EXAMPLE 2 Synthesis of H-D-Ala-Phe-Arg-CHA

I. Z-D-Ala-Phe-Arg(NO₂)-CHA

2.69 Grams (5 m moles) of H-Phe-Arg(NO₂)-CHA.HCl was dissolved in 10 mlof 1.5N-NEM/DMF, to this solution was added 1.73 g (5 m moles) ofZ-D-Ala-SDP at 0° to 5° C., the reacted at room temperature for 18hours. After the reaction was completed, the reaction mixture wasdiluted with 150 ml of AcOEt, the washed 3 times with 50 ml of cold5%-hydrochloric acid and washed twice with 50 ml of an aqueous solutionsaturated with sodium chloride, then decolored and dried with activatedcarbon and anhydrous magnesium sulfate. After the drying, magnesiumsulfate and activated carbon were removed by filtration, and the solventwas removed from the filtrate by evaporation under reduced pressure toobtain crude product of Z-D-Ala-Phe-Arg(NO₂)-CHA, recrystallized fromMeOH/AcOEt/n-hexane to obtain 2.8 g (80.0%) of Z-D-Ala-Phe-Arg(NO₂)-CHA.

Rf₁ =0.91. Melting point: 180°-189° C.

[α]_(D) ²⁰ -5.0 (C=1, DMF).

    ______________________________________                                        Elementary analysis (for C.sub.33 H.sub.38 N.sub.8 O.sub.10.1/2H.sub.2        O)                                                                                       C         H      N                                                 ______________________________________                                        Calculated (%):                                                                            55.38       5.49   15.66                                         Found (%):   55.08       5.69   15.36                                         ______________________________________                                    

II. H-D-Ala-Phe-Arg-CHA.2HCl

2.3 Grams (3.25 m moles) of Z-D-Ala-Phe-Arg(NO₂)-CHA was suspended in amixed solvent of 113 ml of MeOH, 35 ml of H₂ O and 2.1 ml of 1N-HCl. Tothis suspension was added 1 g of palladium black and reduced byhydrogenation at 30° C. for 4 hours. After the hydrogenation wascompleted the catalyst was removed by filtration, further the solventwas removed by evaporation under reduced pressure. The residue thusobtained was purified by using a gel filtration column of TOYOPEARL HW40F with MeOH as the developing solvent to obtain 1.5 g (78.2%) ofH-D-Ala-Phe-Arg-CHA.2HCl.

Rf₃ =0.37. Melting point: 185°-210° C.

[α]_(D) ²⁰ -12.0 (C=0.5, MeOH).

    ______________________________________                                        Elementary analysis (for C.sub.25 H.sub.35 N.sub.7 O.sub.6 Cl.sub.2.3H.sub    .2 O)                                                                                    C         H      N                                                 ______________________________________                                        Calculated (%):                                                                            45.88       6.31   14.98                                         Found (%):   45.41       5.88   15.33                                         ______________________________________                                    

EXAMPLE 3

By a method similar to that described in the previous Examples 1 and 2,there were synthesized substrates as follows:

    ______________________________________                                                          Melting                                                     Substrate of the  point           [α].sub.D.sup.20 (C =                 present invention (°C.)                                                                           Rf.sub.3                                                                             0.5 meOH)                                   ______________________________________                                        PS-2101           188-205  0.39   -24.0                                       H--D-Val--Phe--Arg--CHA                                                       PS-2102           193-206  0.29   -17.0                                       H--D-PGlu--Phe--Arg--CHA                                                      PS-2104           176-198  0.40   -25.0                                       H--D-Phe--Phe--Arg--CHA                                                       PS-2105           185-201  0.40   -24.0                                       H--D-Leu--Phe--Arg--CHA                                                       PS-2106           198-213  0.14   -24.0                                       H--D-Lys--Phe--Arg--CHA                                                       ______________________________________                                        Elementary analysis                                                                             C         H      N                                          ______________________________________                                        PS-2101   Calculated (%):                                                                           46.26     6.32 13.99                                              Found (%):  46.32     6.06 14.09                                    PS-2102   Calculated (%):                                                                           49.93     5.58 15.09                                              Found (%):  50.10     5.74 14.92                                    PS-2104   Calculated (%):                                                                           49.71     5.92 13.09                                              Found (%):  49.94     5.72 13.07                                    PS-2105   Calculated (%):                                                                           47.63     6.42 13.89                                              Found (%):  47.72     6.19 14.16                                    PS-2106   Calculated (%):                                                                           44.95     6.60 14.98                                              Found (%):  45.19     6.18 14.81                                    ______________________________________                                    

EXAMPLE 4

The specificities of newly synthesized substrates were tested byreacting each of them with various enzymes as follows.

(1) Substrate solution: 2 m moles/liter.H₂ O

(2) Buffer solutions: 50 m moles oftris(hydroxymethyl)aminomethane/hydrochloric acid buffer solution and150 m moles/liter of NaCl solution were used, and the reaction pH werespecified with respect to the enzymes as follows.

    ______________________________________                                        Enzymes               pH (at 25° C.)                                   ______________________________________                                        Human kallikrein in urine                                                                       (HUK)   9.0                                                 Thrombin          (TH)    8.5                                                 Plasmin           (PL)    7.8                                                 Urokinase         (UK)    8.2                                                 ______________________________________                                    

(3) Enzymes used:

    ______________________________________                                        Enzyme  Origin  Manufacturer                                                                              Lot No.                                                                              Unit                                       ______________________________________                                        Kallikrein                                                                            Human   The Green   Y1003SM                                                                              0.1  U/ml                                  in urine        Cross                                                                         Corporation                                                   Thrombin                                                                              Bovine  Mochida     65146  4.0  NIH/ml                                                Pharmaceutical                                                                Co., Ltd.                                                     Plasmin Human   The Green   PL-35  0.25 CU/ml                                                 Cross                                                                         Corporation                                                   Urokinase                                                                             Human   Mochida     2C239  1000 U/ml                                                  Pharmaceutical                                                                Co., Ltd.                                                     ______________________________________                                    

(4) Reaction stopper (PNA): 10%-acetic acid

(5) Chromogenic reagent (CHA): Pentacyanoamineferroate

(6) Method of determination:

0.3 Milliliter of the buffer solution and 0.1 ml of the enzyme reagentwere taken in a hard glass test tube of which surface being treated withsilicone, or a plastic test tube and preheated in a thermostat at 37° C.for 5 minutes so as to conduct the enzymatic reaction. Precisely 5minutes after the reaction was started, 2.0 ml of reaction stoppersolution or reaction terminating chromogenic reagent was added toterminate the enzymatic reaction. After the enzymatic reaction wascompleted, the reaction mixture was allowed to stand at 37° C. for 10minutes, and then the absorbance at 405 nm or 700 nm was measured. Theresults are shown in the following Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Comparative test results of substrate specificity                             Substrate              HUK TH  PL  UK                                         __________________________________________________________________________    1. S-2266                                                                            H--D-Val--Leu--Arg--PNA                                                                       0.245                                                                             0.065                                                                             0.067                                                                              0.013                                     2. PS-2103N                                                                          H--D-Ala--Phe--Arg--PNA                                                                       0.109                                                                             0.047                                                                             0.125                                                                              0.006                                     3. PS-2100                                                                           H--D-Pro--Phe--Arg--CHA                                                                       0.244                                                                             0.016                                                                             0.014                                                                              0.003                                     4. PS-2101                                                                           H--D-Val--Phe--Arg--CHA                                                                       0.501                                                                             0.025                                                                             0.025                                                                             0.0                                        5. PS-2102                                                                           H--D-PGlu--Phe--Arg--CHA                                                                      0.128                                                                             0.005                                                                             0.016                                                                             0.0                                        6. PS-2103                                                                           H--D-Ala--Phe--Arg--CHA                                                                       0.599                                                                             0.007                                                                             0.011                                                                             0.0                                        7. PS-2104                                                                           H--D-Phe--Phe--Arg--CHA                                                                       0.172                                                                             0.025                                                                             0.021                                                                             0.0                                        8. PS-2105                                                                           H--D-Leu--Phe--Arg--CHA                                                                       0.144                                                                             0.054                                                                             0.044                                                                             0.0                                        9. PS-2106                                                                           H--D-Lys--Phe--Arg--CHA                                                                       0.193                                                                             0.008                                                                             0.031                                                                             0.0                                        __________________________________________________________________________     The initial substrate concentration S.sub.0 = 0.4 m mole.                     Figures are the measured values of absorbance (O.D.).                         The measurements were conducted at wavelength of 405 nm for the substrate     Nos. 1-2, and at wavelength of 700 nm for the substrates Nos. 3-9.       

What is claimed is:
 1. A composition of matter having the generalformula (I): ##STR3## wherein A₁ is Pro (prolyl group), PGlu(pyroglutamyl group), Val (valyl group), Ala (alanyl group), Leu (leucylgroup), Phe (phenylalanyl group) or Lys (lysyl group) or salt thereof.2. The composition of matter of claim 1 wherein A₁ is a valyl group. 3.The composition of matter of claim 1 wherein A₁ is an alanyl group.
 4. Amethod of determining the activity of kallikrein in the urine whichcomprises reacting a compound having the general formula: ##STR4##wherein A₁ is Pro (prolyl group), PGlu (pyroglutamyl group), Val (valylgroup), Ala (alanyl group), Leu (leucyl group), Phe (phenylalanyl group)or Lys (lysyl group) or salt thereof with urine to form3-carboxy-4-hydroxy aniline; transforming the 3-carboxy-4-hydroxyaniline into a colored substance; and determining the activity of thekallikrein by colorimetric or fluorometric analysis of the3-carboxy-4-hydroxy aniline.
 5. The method of claim 4 wherein A₁ is avalyl group.
 6. The method of claim 4 wherein A₁ is an alanyl group.